Proteases play essential roles in many disease processes such as Alzheimer""s, hypertension, inflammation, apoptosis, and AIDS. Compounds that block or enhance their activity have potential as therapeutic agents. Because the normal substrates of peptidases are linear peptides and because established procedures exist for making non-peptidic analogs, compounds that effect the activity of proteases are natural subjects of combinatorial chemistry. Screening compounds produced by combinatorial chemistry requires convenient enzymatic assays.
The most convenient existing assays for proteases are based on fluorescence resonance energy transfer from a donor fluorophore to a quencher placed at opposite ends of a short peptide chain containing the potential cleavage site. Knight CG, xe2x80x9cFluorimetric assays of proteolytic enzymes,xe2x80x9d Methods in Enzymol. (1995) 248:18-34. Proteolysis separates the fluorophore and quencher, resulting in increased intensity in the emission of the donor fluorophore. Existing protease assays use short peptide substrates incorporating unnatural chromophoric amino acids, assembled by solid phase peptide synthesis. However, solid phase synthesis poses certain problems of effort and expense.
It is useful to perform enzymatic assays in vivo, in order to more closely mimic conditions in which intracellular proteases act. Conventional artificial substrates prepared by solid-phase synthesis would require microinjection into individual cells, which is impractical as a high-throughput screen. Also, short unfolded peptides are generally rapidly degraded by nonspecific mechanisms inside cells.
The Edans fluorophore is the current mainstay of existing fluorometric assays. Fluorophores with greater extinction coefficients and quantum yields are desirable. The Edans fluorophore often is coupled with a non-fluorescent quencher such as Dabcyl. However, assays performed with such agents rely on the absolute measurement of fluorescence from the donor. This amount is contaminated by other factors including turbidity or background absorbances of the sample, fluctuations in the excitation intensity, and variations in the absolute amount of substrate.
This invention provides tandem fluorescent protein constructs and methods for using them in enzymatic assays both in vitro and in vivo. Tandem fluorescent protein constructs comprise a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a linker moiety that couples the donor and acceptor moieties, wherein the donor and acceptor moieties exhibit fluorescence resonance energy transfer when the donor moiety is excited. The fluorescent protein moieties can be Aequorea-related fluorescent protein moieties, such as green fluorescent protein and blue fluorescent protein. In one aspect, the linker moiety comprises a cleavage recognition site for an enzyme, and is, preferably, a peptide of between 5 and 50 amino acids. In one embodiment, the construct is a fusion protein in which the donor moiety, the peptide moiety and the acceptor moiety are part of a single polypeptide.
This invention also provides recombinant nucleic acids coding for expression of tandem fluorescent protein constructs in which a donor fluorescent protein moiety, an acceptor fluorescent protein moiety and a peptide linker moiety are encoded in a single polypeptide. The invention also provides expression vectors comprising expression control sequences operatively linked to a recombinant nucleic acid coding for the expression of a tandem fluorescent protein construct, as well as host cells transfected with those expression vectors.
The tandem constructs of this invention are useful in assays for determining whether a sample contains an enzyme. The methods involve contacting the sample with a tandem fluorescent protein construct. The donor moiety is excited. Then the degree of fluorescence resonance energy transfer in the sample is determined. A degree of fluorescence resonance energy transfer that is lower than an expected amount indicates the presence of an enzyme. The degree of fluorescence resonance energy transfer in the sample can be determined as a function of the amount of fluorescence from the donor moiety, the amount of fluorescence from the acceptor donor moiety, the ratio of the amount of fluorescence from the donor moiety to the amount of fluorescence from the acceptor moiety or the excitation state lifetime of the donor moiety.
The assay also is useful for determining the amount of enzyme in a sample by determining the degree of fluorescence resonance energy transfer at a first and second time after contact between the enzyme and the tandem construct, and determining the difference in the degree of fluorescence resonance energy transfer. The difference in the degree of fluorescence resonance energy transfer reflects the amount of enzyme in the sample.
The invention also provides methods for determining the amount of activity of an enzyme in a cell. The methods involve providing a cell that expresses a tandem fluorescent protein construct, for example by transfecting the cell with an appropriate expression vector. The cell is exposed to light in order to excite the donor moiety. Then the degree of fluorescence resonance energy transfer in the cell is determined. The degree of fluorescence resonance energy transfer reflects to the amount of enzyme activity in the cell.
Similarly, the invention provides methods of determining the amount of activity of an enzyme in a sample from an organism. The methods involve providing a sample from an organism having a cell that expresses a tandem fluorescent protein construct. The donor moiety in the sample is excited. Then the degree of fluorescence resonance energy transfer in the sample is determined. The degree of fluorescence resonance energy transfer reflects the amount of enzyme activity in the cell.
The assay methods also can be used to determine whether a compound alters the activity of an enzyme, i.e., screening assays. The methods involve contacting a sample containing an amount of the enzyme with the compound and with a tandem fluorescent protein construct; exciting the donor moiety; determining the amount of enzyme activity in the sample as a function of the degree of fluorescence resonance energy transfer in the sample; and comparing the amount of activity in the sample with a standard activity for the same amount of the enzyme. A difference between the amount of enzyme activity in the sample and the standard activity indicates that the compound alters the activity of the enzyme.
Similar methods, are useful for determining whether a compound alters the activity of an enzyme in a cell. The methods involve providing first and second cells that express a tandem fluorescent protein construct; contacting the first cell with an amount of the compound; contacting the second cell with a different amount of the compound; exciting the donor moiety in the first and second cell; determining the degree of fluorescence resonance energy transfer in the first and second cells; and comparing the degree of fluorescence resonance energy transfer in the first and second cells. A difference in the degree of fluorescence resonance energy transfer indicates that the compound alters the activity of the enzyme.